Mu-opioid receptors: A non-synonymous A118G SNP in the human OPRM1 gene that encodes the mu-opioid receptor may modulate alcohol reward and therapeutic response to naltrexone (NTX). We previously created two lines of humanized mice carrying the respective human allele (118AA and 118GG), and observed a markedly enhanced dopamine (DA) release in the nucleus accumbens (NAc) of 118GG mice in response to alcohol. This finding was consistent with a positron emission tomography study conducted in parallel in humans (Ramchandani et al., 2011). More recent work revealed that the enhanced alcohol reward in the 118GG mouse as measured by alcohol-induced decrease in intracranial brain stimulation (ICSS) threshold. The ICSS threshold suppression is selectively blocked by the opioid antagonist naltrexone, and approved alcoholism medication (collaboration with Robinson and Malanga, UNC). Accordingly, voluntary alcohol consumption in 118GG mice is increased compared to the 118AA line, and the 118GG mice are preferentially sensitive to naltrexone (Bilbao et al., Biol Psychiat, in press). NK1 receptors: We previously found that L822429, a rat-specific NK1 antagonist dose-dependently suppressed stress-induced reinstatement of alcohol seeking in Wistar rats, but had no effect on cue-induced reinstatement (Schank et al., 2011). In follow-up work,, L-822429 dose-dependently suppressed self-administration in alcohol preferring P-rats but was ineffective in non-dependent Wistar rats. We found an up regulation of NK1R expression in the central amygdata (CeA) of P-rats; microinjections of the antagonist into this region mimicked systemic effects (Schank et al., 2013). Fos-mapping showed that stress-induced reinstatement of alcohol seeking is associated with neuronal activation in the amygdala (amg), NAc, dorsal raphe nucleus (DR), medial prefrontal cortex (mPFC) and bed nucleus of the stria terminalis. NK1R antagonism suppressed the stress-induced increase in Fos in a subset of these regions, particularly the DR and NAc shell, suggesting that these regions mediate the effect of NK1R antagonism to block stress-induced relapse. Furthermore, we identified that NK-1 antagonism attenuates yohimbine-induced reinstatement of alcohol seeking and cocaine seeking (Shank et al., 2014). Neuropeptide S (NPS) receptors: NPS signaling may play a role in relapse to alcohol seeking (Canella et al., 2009). In collaboration with the NIH Chemical Genomics Center (NCGC), we identified a lead molecule, NCG001865684, that is brain penetrant upon peripheral administration in rats. In vitro, NCGC00185684 shows biased antagonist properties, and preferentially blocks ERK-phosphorylation over intracellular cAMP- or calcium-responses to NPS. In vivo, pretreatment with NCG001865684 blocks alcohol-induced ERK-phosphorylation in the rat CeA, a region involved in regulation of alcohol intake. NCGC00185684 also decreases operant alcohol self-administration, and lowers alcohol reward as measured using progressive ratio responding. These effects are behaviorally specific. Taken together, these data provide an initial validation of the NPSR as a therapeutic target in alcoholism (Thorsell et al., 2013). Melanin Concentrating Hormone (MCH): MCH is a hypothalamic appetite regulating peptide that is also involved in reward, motivation and anxiety-like behaviors mediated by the MCH-1 receptor (MCH-1R) in rodents. MCH1-R blockade suppresses alcohol self-administration (Cippitelli et al., 2010). Because alcohol is also a caloric nutrient, self-administration results could be confounded by appetite effects. We have therefore evaluated the role of the MCH system for the rewarding properties of alcohol using conditioned place preference (CPP) in MCH-1R knockout (KO) mice. In contrast to wildtype C57BL/6 mice, MCH-1R KO:s did not develop place preference for at a 2g/kg dose. This effect was behaviorally specific, as no differences were observed on locomotion, loss of righting reflex (LORR), or alcohol metabolism and elimination (Karlsson et al., in prep). Proinflammatory activity: Innate immunity might be involved in stress responses and behaviors related to addiction. We began investigating the role of the IL-1 receptor (IL-1R) in alcohol related behaviors. Mice lacking IL-1Rs consumed less alcohol in a two bottle choice test, but CPP experiments showed that the decreased alcohol consumption in the IL-1R mice is not due to effects on alcohol reward. IL1-R KO mice had significantly longer sleep time when evaluated for LORR, indicating higher sensitivity to sedative / ataxic properties of alcohol than controls. This is in agreement with a frequently observed inverse correlation between sensitivity to sedative / ataxic effects of alcohol and consumption. In a model of stress-induced drinking, social defeat stress (SDS), both genotypes potently increased their consumption acutely, but no genotype differences were seen. Because IL-1 beta and TNF-a share overlapping pathways, it has been suggested that they can act in parallel, and can substitute for each other. We next therefore used a double KO mouse model lacking both IL-1 and TNF-1 receptors. The double KOs consumed less alcohol, and did not escalate their consumption with increasing alcohol concentrations. This is not likely to be due to differences in the rewarding properties of alcohol. Double KO mice show a robust expression of alcohol induced CPP. Further Double KO mice were assessed on LORR, alcohol metabolism and taste preference. No differences were observed suggesting behaviorally specific effects. Interestingly, double-KO mice are less sensitive to stress induced drinking after SDS exposure and did not increase their consumption from baseline intake. (Karlsson et al., in prep). DNA methylation: Substantial evidence supports a role of epigenetic mechanisms in the regulation of alcohol-related behaviors. We found that i.c.v. infusion of RG-108, a specific DNA methyltransferase inhibitor, decreased operant alcohol self-administration in post-dependent rats, and reversed long-term expression changes encoding synaptic proteins involved in neurotransmitter release in the mPFC induced by a history of dependence. The effects of RG-108 were reproduced with local infusion into the mPFC over 7-10 days and were behaviorally selective. Infusion of RG108 prevented both escalation of alcohol consumption and dependence-induced down-regulation of 4 out of the 7 transcripts modified in PD rats. Specifically, RG108 treatment directly reversed the down-regulation of synaptotagmin 2 (syt2), which was caused by alcohol-induced hypermethylation. Lentiviral inhibition of Syt2 expression in the mPFC increased aversion-resistant alcohol drinking, supporting a mechanistic role of Syt2 in alcohol seeking. (Barbier et al., in submission). microRNA regulation: Following up on a recent microarray screen (Tapocik et al., 2012), we qPCR confirmed that alcohol dependence results in persistent up-regulation of miR-206 in the mPFC. Overexpression of miR-206 in the mPFC of non-dependent rats reproduced the escalation of alcohol self-administration. Accordingly, BDNF was downregulated in PD rats on microarray analysis, and this was confirmed by qPCR. In vitro, BDNF expression was repressed by miR-206 in a 3'UTR reporter assay, confirming BDNF as a functional target of miR-206. Inhibition of miR-206 expression in differentiated rat cortical primary neurons significantly increased secreted levels of BDNF. Therefore, recruitment of miR-206 in the mPFC contributes to escalated alcohol consumption and BDNF regulation (Tapocik et al, 2014). We have recently confirmed this finding in a mouse model of alcohol dependence where BDNF expression is down and mir-206 expression is upregulated. Furthermore, miR-206 KO mice have a blunted ethanol response to alcohol vapor (Tapocik et al.,in prep).